Hrp conjugated anti rabbit igg

Whole-cell extracts were prepared from the DT40 cell lines, and were separated by SDS-PAGE. The gels were transferred onto PVDF membranes (FluoroTrans, PALL). The membranes were blocked with 5% skim milk (BD), and then probed with the following antibodies: rabbit anti-GFP (1:5000; MBL), rabbit anti-chicken CENP-A (1:5000)^{73 (link)}, mouse anti-H4K20 monomethylation (1:5000; #CMA421)^{28 (link),49 (link)}, or Rat anti-pan H3 (1:5000; #140-1G1)^{74 (link)} as the primary antibody. HRP-conjugated anti-rabbit IgG (1:15,000; Jackson ImmunoResearch; 111-035-144) and HRP-conjugated anti-mouse IgG (1:15,000; Jackson ImmunoResearch; 115-035-003) were used as secondary antibodies. Signals were developed using ECL Prime (GE Healthcare), and were detected by a ChemiDoc Touch imaging system (Bio-Rad). The uncropped images of all blots are presented in the Supplementary Figures 12 and 13.

Goat polyclonal anti-PCSK9 from R&D (Minneapolis, MN, United States), rabbit polyclonal anti-PCSK9 from Abclonal Science Inc. (Woburn, MA, United States), mouse monoclonal anti-IR-β subunit from Millipore (Etobicoke, ON, Canada), rabbit polyclonal anti-IR-α subunit from Biorbyt (Cambridge, United Kingdom); rabbit polyclonal anti LDL-R, rabbit polyclonal anti-ABCA1, purified rabbit polyclonal anti-SR-BI, rabbit polyclonal anti-SR-BII, rabbit polyclonal anti-IL-17 and rabbit polyclonal anti-LDL-R from Novus Biologicals (Littleton, CO, United States); rabbit polyclonal anti-ACAT-1 and anti-ACAT-2 from Cayman Chemical (Ann Arbor, MI); chicken polyclonal anti-hormone-sensitive lipase (HSL) from ProSci Inc. (Poway, CA, United States); and rabbit polyclonal anti-IL-17RA from Abcam (Cambridge, MA, United States); HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-goat IgG, biotinylated anti-rabbit IgG from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, United States); HRP-conjugated anti-chicken IgG (H + L) made in goat from Vector Laboratories Inc. (Burlingame, CA, United States).

One million transfected HEK-293 cells were plated in 1 ml for 24h and gp96-Ig levels were determined in the supernatant by ELISA using 10 μg/ml goat anti-human IgG antibody (Jackson ImmunoResearch) for detection and human IgG1 Kappa (Jackson ImmunoResearch) as a standard (Figure 1B). Protein expression was verified by SDS-PAGE and Western blotting using rabbit anti-P. falciparum CSP protein antibody (CSP 207-397, Alpha Diagnostic International) at 1/1000 dilution and rabbit anti-P. falciparum AMA-1 protein (Cusabio) at 1/200 dilution. Binding of the primary antibodies was detected with HRP conjugated anti rabbit IgG (Jackson ImmunoResearch) at 1/5000 dilution. Protein bands were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) (Figure 1C).

Control (20 nM) or miR-4669 mimic (10 or 20 nM)-transfected Hep3B cells were cultured for 72h, and GAPDH levels in the culture medium were semi-quantified using western blot analysis. Briefly, proteins in culture media (10 μL) were fractionated and transferred onto a PVDF membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The membrane was immunoprobed with a rabbit anti-GAPDH monoclonal antibody (×500 dilution, Abcam, Cambridge, UK) followed by incubation with HRP-conjugated anti-rabbit IgG (×5000 dilution, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Signals were visualized using an Immunobilon Forte Western HRP substrate (MilliporeSigma), and signal intensity was quantified using a G:BOX Image Station iChemi XL device (SYNGENE, Cambridge, UK).

Immunostaining of glycolipids on TLC sheets was performed according to the method described by Watarai et al. [23 (link)], with certain modifications. Glycolipids (1 μg) were spotted onto TLC silica gel polyester sheets (100 × 100 mm, Polygram, Macherey-Nagel, Germany) and developed with chloroform/methanol/water/acetic acid (60:40:6:0.2 vol.) at room temperature for 30 min and then air-dried. Glycolipids on the TLC sheets were visualized with a sprayed 5-methylresorcinol (Orcinol, Tokyo Chemical Industry, Tokyo, Japan) solution (2 mg/mL in 2 M H₂SO₄) and heated at 110°C for 5 min. For immunostaining, TLC sheets were immersed in PBS containing 1% gelatin, 1% polyvinylpyrrolidone (PVP-K30, Nacalai Tesque, Kyoto, Japan), and 1 mM EDTA, followed by immersion in anti-ASGM1 rabbit mAbs (50 ng/mL) or PoAb (1:1,000) solution and allowed to react for 1 h at room temperature. After washing thrice with T-PBS, HRP-conjugated anti-rabbit IgG (1:40,000; The Jackson Laboratory, Bar Harbor, ME, USA) was added. After washing thrice with T-PBS, the TLC sheets were incubated in TMB solution for western blotting (Nacalai Tesque, Kyoto, Japan). Consequently, the bound antibody was visualized in blue.

BHK-21 cells were inoculated with 0.01MOI BEFV after transfection with 50 nM of miR-3470b mimic, NC mimic, miR-3470b inhibitor and NC inhibitor respectively at the indicated time points. After three times freeze-thaw, cells and culture medium were harvested for virus titration expressed as the lgTCID₅₀/ml by the Reed-Muench endpoint method. Meanwhile, BEFV N gene mRNA levels were determined by real-time RT-PCR assay [39 (link)]. All samples were run in triplicate and fold changes were calculated using the 2^-ΔΔCt method. What’s more, BEFV G protein levels were analyzed by western blot as described above using the prepared rabbit anti-G1 polyclonal antibodies with 1/128 virus neutralization titer. Briefly, BEFV infected cells were lysed, and total proteins were subjected to western blot probed with rabbit anti-G1 polyclonal antibodies at a 1: 500 dilution or an anti-β-actin antibody at a 1: 5000 dilution (Abcam, USA). Then, HRP-conjugated anti-rabbit IgG (Jackson, USA) at a 1:3000 dilution was used to label primary antibody. Immunolabelled proteins were visualized using ECL reagent (Pierce).

Due to unique cellular responses observed in HFLS-RA cells (compared to HFLS-OA or HFLS control cells), cell signaling pathways were examined only in HFLS-RA cells. Several signal transduction pathways involved in the transformation of HFLS-RA cells to an aggressive phenotype were examined (16 (link), 50 (link), 59 (link)). Briefly, HFLS cells were incubated with antigens for 15-min, 30-min, 1-h and 4-h, cellular lysates were prepared as above, and the following primary antibodies (all polyclonal rabbit IgGs) were utilized to probe the samples; anti-β-actin antibody (endogenous control) (Novus Biologicals), anti-SAPK/JNK (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-Erk1/2 (p44/42 MAPK) (#9102), anti-phospho-Erk1/2 (p44/42 MAPK; Thr202/Tyr204) (#9101), anti-Akt (#9272), anti-phospho-Akt (Ser473) (#9271), anti-p38 MAPK (#9212), anti-phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-Paxillin (#2542), anti-phospho-Paxillin (Tyr118) (#2541), anti-FAK (#3285), anti-phospho-FAK (Tyr397) (#3283) (Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch) was used as a secondary antibody. Respective band intensities were measured and reported as outlined above. The maximum and minimum activation of signaling pathways for HFLS lysates was determined based on positive (treatment with LPS) and negative (treatment with media) controls.